Bacterial and synthetic polysaccharide immunomodulators that enhance general immunity

ABSTRACT

The present invention relates to the effects of bacterial polysaccharides on cell mediated immunity in animals. Polysaccharides of the present invention comprise the outer polysaccharides (OPS) located on bacterial cell membranes and other polysaccharides (e.g. “Poly B”) either secreted or contained within the periplasmic space. These polysaccharides have been found to enhance the general or cell mediated immunity of animals to various diseases. The invention provides for the use of such polysaccharides in preventing and treating various infections as well as in treating carcinomas. The invention also provides for synthetic polysaccharides having the same immuno-modulating effect as the bacterial polysaccharides.

The present application is based on, and claims benefit of, U.S.Provisional Application No. 60/021,112, filed, Jul. 3, 1996.

BACKGROUND OF THE INVENTION

Infectious agents, parasitic diseases or cellular pathologies arebecoming more resistant to conventional methods of protection ortreatment. These methods include vaccines, antibiotics, orimmuno-modulating drugs. In controlling disease, one field that isgaining momentum is the development of compounds that stimulate thebody's natural defences. These natural defences have been simplified asbeing of two responses: humoral immunity and cell mediated immunity.

Humoral, or specific, immunity involves the production of specificantibodies in an infected animal as a result of the presence of aninvading microorganism or toxin having a particular antigen. Theseantibodies either congeal or inactivate the invader, or interact withother blood components, such as complement (a system of plasma proteinsthat interact with antibodies), to destroy it.

Cell mediated (cellular) immunity involves, in part, the activation ofphagocytic cells in the host animal to become metabolically more activeand digest or chemically neutralize invading foreign objects.

There are difficulties in stimulating either of these defences. In thecase of humoral immunity: it requires several months of immunization todevelop a response; antibody levels resulting from this form of immunitydiminishes over several months; not all vaccines are effective intriggering an immune response (e.g. susceptibility to Dengue Feveractually increases with experimental vaccination); humoral immunity isuseless against parasitic diseases (which invade the cells and areprotected from antibodies or drugs); and, it is very specific to theserotype of a given microbe.

With cell meditated immunity, “immuno-modulators”, or drugs thatstimulate activity, enhance cellular activity either poorly or withlittle effect. Further, these compounds are frequently toxic (resultingin nausea, fevers, malaise and death), and may have unwelcome sideeffects on the activity of other cell sub-populations. Examples of suchimmuno-modulators are nucleic acid analogues, chemically defined drugs,adjuvants and biologically active peptides. These drugs are useful foronly certain diseases.

One group of immuno-modulators that appears to have become overshadowedby other modulators are glycans which are carbohydrates extracted frommicro-organisms, especially plants and yeast (Seljelid et al, 1981,“Glycan Stimulation of Macrophages in vitro”, Exp. Cell. Res.131:121-129). Glycans are also referred to as glucans, but in thisreport the term glucans will be used to refer to polymers of glucose.Seljelid et al (1981) found that “only insoluble glycans are effectivein stimulating macrophages” and, of these glycans, the presence of1,3-beta linkages of the sugars was believed to be a contributingfactor.

D'Hinterland et al in U.S. Pat. No. 4,734,403 (issued Mar. 29, 1988)isolated a membrane polysaccharide from bacteria which was found to havean immuno-modulating effect on natural killer cells to destroy Maloney'slymphoma. The polysaccharide taught in this patent comprises a chain ofgalactofuranose and galactopyranose units and is isolated from gramnegative bacteria. In U.S. Pat. Nos. 4,933,440 and 4,937,327,d'Hinterland et al teach immuno-modulators derived from the bacterialpolysaccharides recited in the 4,734,403 patent.

The polysaccharide noted by d'Hinterland et al is extracted by cellulardisruption, alkaline digestion and dissolving in water; it is unknownwhether this polysaccharide can protect against its source bacterium,Klebsiellapneumoniae. As most immuno-modulators act only for a few days,this may be why the in vivo trials disclosed in the d'Hinderlandreferences were conducted at most 3 days after immunization. Thepolysaccharide had adjuvant properties for enhancing antibodies (i.e.humoral immunity not cellular immunity) when it was given at the sametime as a ribosomal vaccine. These publications are encouraging forother researchers, not only because of the anti-cancer properties ofd'Hinderland's discovery, but also because of their findings thatbacterial polysaccharides may have immunological effects.

In contrast, the present inventors have discovered that theO-polysaccharide (OPS) of Brucella used previously to eitherdifferentiate infected from vaccinated cattle (Cherwonogrodzky et al.,U.S. Pat. No. 5,006,463) or to protect animals from brucellosis(Cherwonogrodzky et al., Canadian application 2,164,155), hasimmunomodulating properties. Although this polysaccharide is associatedwith the cell membrane when bound to lipopolysaccharide or LPS, instrain B. melitensis B 155 it is found loose in the periplasmic space,in B. melitensis 16M it is shed into the medium, and there is stillcontroversy if it can form a capsule or form “native hapten” which issecreted away from the cell. Also, another polysaccharide associatedwith Brucella but not found in many other bacteria, referred to hereinas “poly B”, has similar immunomodulating effects in that it can alsogreatly increase the activity of macrophages as evidenced bychemilumenescence. These polysaccharides differ in composition,structure and sometimes location to the polysaccharide noted byd'Hinterland and these are purified by different methods. Thesepolysaccharides (there is more known about the OPS) appear to have somedifferent properties compared to the one presented by d'Hinterland.

SUMMARY OF THE INVENTION

Accordingly, in one embodiment, the present invention provides animmuno-modulating compound comprising an effective, non-toxic quantityof bacterial polysaccharide for enhancing cell mediated immunity in ananimal against bacteria, fungi, yeast, viruses, parasites and cellularabnormalities.

The polysaccharides of the invention comprise bacterial OPS and Poly Bmolecules and polysaccharides cross reactive therewith and mixturesthereof.

Further, the invention provides for the use of such polysaccharides inthe treatment and prevention of various infections as well as thetreatment of carcinomas and other cellular pathologies.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the preferred embodiments of the inventionwill become more apparent in the following detailed description in whichreference is made to the appended drawings wherein:

FIG. 1 is a graph showing the effect of Brucella abortus0-polysaccharide (OPS) for enhancing neutrophil/macrophage activity ofBalb/c mice as determined by chemiluminescence assay.

FIG. 2 is a graph showing the effect of Yersinia enterocolitica 0-90-polysaccharide (OPS) for enhancing neutrophil/macrophage activity ofBalb/c mice as determined by chemiluminescence assay.

FIG. 3 is a graph showing the effect of Agrobacterium tumefaciens1,2-beta linked glucose polymer for enhancing neutrophil/macrophageactivity of Balb/c mice as determined by chemiluminescence assay.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Although bacterial polysaccharides have been used as vaccines to enhancespecific humoral immunity (i.e. to increase antibodies in the bloodtowards a specific antigen), the present invention uses such compoundsto enhance general cellular immunity against a wide variety ofmicroorganisms.

The inventors have found that soluble carbohydrate polymers, extractedfrom bacteria, can serve as potent immuno-modulators, stimulating thedefences of white blood cells. In this investigation, theimmuno-modulating effects of OPS (outer polysaccharide) from B. abortuswas studied along with that from Yersinia enterocolitica O:9, abacterium unrelated to the former but which synthesizes a similar OPS.

A study of the immunological effects of Brucella OPS found it to be aneffective vaccine for preventing brucellosis in swine. A description ofthis study is found in Applicant's co-pending Canadian patentapplication number 2,164,155, filed Nov. 30, 1995. From the results ofthis study, it was also found that the OPS from Brucella also hadimmuno-modulating effects. The observations leading to this finding wereas follows:

1. The antibody response was feeble to non-existent, as determined bymeasuring titres of IgG and IgM specific to OPS.

2. Where antibodies were induced either with the use of adjuvantsliposomal encapsulation, high doses and/or multiple injections of thiscomponent, protection from infection was improved when the humoralimmunity was reduced. An example of such adjuvants is Lipid A bound toOPS when smooth-lipopolysaccharide, or sLPS, is used.

3. The immunity that resulted from this vaccine seemed generic ratherthan specific. Not only did it protect animals from different species ofBrucella (e.g. mice and guinea pigs from B. abortus, swine from B.suis), but it also protected swine from an outbreak of Haemophiluspleuropneumoniae.

4. The OPS was exceptionally potent and long lasting. 100 μg of the OPSprotected a sow that was immunized at 2 months of age and 25 kg inweight. Such protection was evident when the sows were challenged 6months later and continued one year later. During this time antibodytitres were not detectable.

Further, an investigation was made of the immuno-modulating effects of aglucose polymer, referred to as “Polysaccharide B” or “Poly B,”synthesized by Brucella abortus. Poly B is one of a number of unusualsugars (i.e of unusual conformation and/or linkages) produced byBrucella as well as several other bacteria (including plant pathogenssuch as Agrobacterium tumefaciens) and is a circular molecule comprisinga ring of 1,2-linked-β-D-glucose. This compound, whose function isunknown, cannot be digested and has not been found to induce an antibodyresponse; however, it has been found to be a powerful immuno-modulator.To avoid the possibility of the immuno-modulating activity being causedby contamination by Brucella OPS, the source of this cyclic glucan(glucose polymer) was Agrobacterium tumefaciens.

The Brucella OPS comprises a polymer of4-formamido-4,6-dideoxy-D-mannose having a molecular weight of about15,000 to 18,000. Specifically, this OPS is composed of a repeatingpentasaccharide unit containing a sequence of one 1,3- and four1,2-linked 4,6-dideoxy-4-formamido-α-D-mannopyranosyl units. The Poly Bmolecule has been identified as a family of circular 1,2- linkedpolymers of β-D-glucopyranosyl units ranging in ring size from 17 to 24glucosyl units (Bundle et al, 1987, “The Structure of theLipopolysaccharide O-Chain (M Antigen) and Polysaccharide B Produced byBrucella melitensis 16M”, FEBS Lett., 216:, 261-264). Further, the PolyB used in the present study is not located on the cell membrane but inthe periplasmic space.

Evidence from swine trials supports the view that theseimmuno-modulators can protect against a broad range of infections (e.g.different Brucella species, Haemophilus pleuropneumoniae). It is alsoexpected that these immuno-modulators can also act as therapeutic agentsfollowing infection. Unlike the view expressed by Seljelid et al.(1981), the O-polysaccharides of either B. abortus or Y enterocoliticaO:9 are polymers of 4-formamido-4,6-dideoxy-D-mannose with 1,2 alpha and1,3 alpha (not beta) linkages (Caroff et al, 1984, “Structure of theO-Chain of the Phenol Phase Soluble Cellular Lipopolysaccharide ofYersinia enterocolitica Serotype O:9”, Eur. J. Biochem., 139, 195-200;Caroff et al, 1984, “Antigenic S-Type Lipopolysaccharide of Brucellaabortus 1119-3”, Infect. Immun. 46, 384-388; Cherwonogrodzky, 1995, “APolysaccharide Vaccine to Enhance Immunity Against Brucellosis”, Arch.Med. Vet., 27, 29-37).

There is anecdotal evidence that these polysaccharides can enhance theimmune response to vaccines against viral diseases, even when givenbefore vaccination. When investigators were immunized against WesternEquine Encephalitis (WEE), the average titre was 103±117. Aninvestigator working on these noted polysaccharides (and who was likelyto have been exposed to trace amounts of these polysaccharides prior toimmunization), following WEE vaccination, had titres greater than10,000.

Of the several thousands of bacterial OPS's available for study, theabove findings make it logical to begin with the OPS of Brucella speciesthat have shown positive results, then to progress to the OPS ofbacteria that cross-react with Brucella (e.g. the OPS of Y.enterocolitica O:9), and then investigate other bacterial OPS, such asPoly B for immuno-modulating effects. OPS and Poly B may appear to bevery different sugars, but these were tested because these both haveunusual compositions or structures. By unusual it is meant that suchcompositions or structures are unlikely to be a part of normal mammalianmetabolism. Indeed, by being unusual these sugars may not be metabolizednormally and hence act as metabolic signals for cellular immunity. OPSis a formamido sugar with 1,2 and 1,3α linkages and Poly B is 1,2βlinked glucose in a circle.

MATERIALS AND METHODS Preparation of Bacterial Compounds

Brucella abortus strain 413 was acquired from Agriculture Canada, AnimalDiseases Research Institute (ADRI-Nepean), Nepean, Ontario, Canada.Bacteria were grown in Brucella broth (Difco/BDH Inc., Edmonton,Alberta) and incubated with 5% CO₂ at 37° C. for a week. The cells werekilled with 2% phenol. To extract OPS, the method of Cherwonogrodzky etal. (“Antigens of Brucella, Animal Brucellosis, K. Nielsen and J. R.Duncan(eds.), CRC Press, Boca Raton, 19-64, 1990) was used. Briefly, thekilled cells were suspended in 2% acetic acid in 1% saline (thesuspension was 20% cells, v/v), placed in a boiling water bath for 2hours, centrifuged to remove cells, trichloroacetic acid (finalconcentration of 0.2 M) was added to remove proteins, this wascentrifuged and the supernatant was extracted at room temperature withan equal volume of phenol. The OPS was precipitated from the phenollayer with 5 volumes of methanol with 1% sodium acetate (w/v), dialyzedagainst 0.4% acetic acid/0.4% pyridine as buffer, then purified on aG-50 Sephadex™ column with this same buffer. Eluted OPS was lyophilizedand kept as a dry powder until required.

The OPS of Yersinia enterocolitica O:9 was purified by the method ofCaroff et al. (1984) which is similar to that noted above.

Agrobacterium tumefaciens 15955 was acquired from the American TypeCulture Collection (ATCC) (Rockville, MD). It was grown in 4 liters of1.5% glucose, 0.2% calcium carbonate supplemented with about 0.5% yeastextract of less than 8000 m.w. (40 grams of yeast extract was suspendedin about 200 ml of distilled water, dialyzed and the contents outsidethe dialysis bag was used for the media) for 4 days at room temperaturewith constant stirring. At the end of this incubation, the culture waskilled with 2% phenol, cells were removed with centrifugation, and theglucans were precipitated from the supernatant with 4 volumes ofmethanol with 1% sodium acetate. The precipitate was collected bycentrifugation and, after discarding the supernatant, dissolved anddialyzed against distilled water, then lyophilized and kept as a drypowder until required.

Swine Trials

0.1 to 0.5 mg of B. abortus or B. suis OPS was dissolved in 1 ml ofsterile 0.85% saline and injected into the jugular veins of sows.Alternatively, 0.1 to 0.5 mg of OPS was packed into a hole drilled intoa pecan nut and fed to the swine. Sets of 10 swine were given eithersingle or multiple (3 doses) inoculations. Swine were immunized at 2months of age when they were about 25 kg, and then, 6 months later, weremated with 4 boars that were infected and shedding B. suis in the semen.A year later the sows were mated with infected boars again to determineif the vaccine had long lasting protection.

Measuring Enhanced White Blood Cell Activity

To measure immuno-modulation, a microplate chemiluminescent assay wasused, based on the method of Kournikakis and Simpson (“Optimization of aPhagocyte Microplate Chemiluminescent Assay”, 1995, J Biolumin.Chemilumin., 10). In brief, Balb/c mice (15-16 grams, 29-35 day oldfemales) were purchased from Charles River, St. Constant, Quebec. Setsof 5 mice were injected with bacterial compounds in phosphate bufferedsaline (PBS), or as a control only PBS was used, intraperitoneally in1.0 ml. Seven days later, the mice were sacrificed by cervicaldislocation. 5 ml of Hank's balanced salt solution without sodiumbicarbonate (HBSS-) (Gibco laboratories, Grand Island, N.Y.) wasinjected into the abdomen with a syringe having a 22 gauge, 1 inchangiocatheter attached and this was inserted into the peritoneal cavity.The abdomen was then massaged for 3 minutes, and the macrophagesuspension was drawn off into the syringe. This was repeated twice moreand all suspensions for a set of mice were pooled and diluted to a cellconcentration of 1.0×10 cells/ml in Hank's balanced salt solution withCaCl₂ (HBSS+). The pooled suspension was centrifuged at 250 g for 10min. The resulting pellet was re-suspended in 7 ml HBSS- and layeredover 3 ml Histopaque™ 1083 (Sigma Chemical Col., Mo.). This wascentrifuged at 700 g for 35 minutes. The cell layer was then collectedand washed two times in HBSS-.

Phagocytic activity of macrophages was judged by enhanced luminescenceof cells as a result of ingesting Lucigenin™ (Sigma Chemical Co., St.Louis, Mo.) when the neutrophils/monocytes phagocytized zymosanparticles. These particles were prepared by boiling Zymosan A™, SigmaChemical Co., collecting the insoluble particles and opsonizing withantibodies by incubating with human serum; see Kournikakis and Simpson,1995). 100 μL of lucigenin™ was added to each well of a Titertek™fluoroplate. 50 μL of the white blood cell suspension was added andafter an incubation of 37° C. for 10 min, the Luminoskan™ controlprogram added 100 μL of zymosan particles automatically. Luminescencewas detected and recorded automatically by the Titertek™ Luminoskan™(ICN Biomedicals, Inc., Huntsville, Ala.).

Western Equine Encephalitis Vaccination

Seven researchers were immunized with the Western Equine Encephalitisvaccine (formaldehyde killed strain CM-4884, produced by the SalkInstitute, Swiftwater, Pa. USA). Administration was 3 subcutaneousdoses, each given months apart. The vaccinated researchers were noted asbelonging to either of 2 groups: six had good titres to the vaccine andhad not worked with the polysaccharides noted in this invention, one hadextraordinarily high titres and did work with these polysaccharides.

Results

1. Animal Studies

The use of B. abortus OPS to protect mice from brucellosis has beenreported (Cherwonogrodzky et al, 1995) as has its use to protect guineapigs and swine (Cherwonogrodzky et al., 1996, “A Polysaccharide Vaccineto Enhance Immunity Against Brucellla Species, 7^(th) Intl. Conf. forInfec. Dis., Hong Kong, abstract 11029). For the latter, swine given 0.1mg to 0.5 mg, whether as a single dose or three doses each 1 week apart,or given intravenously or orally, were protected from brucellosis 6months after immunization. One year later, the swine were stillprotected from brucellosis. Two months later, vaccinated swine wereprotected from an outbreak of Haemophilus pleuropneumoniae which eithermade ill or killed unvaccinated control swine.

2. Immuno-Modulation of Mice

Immuno-modulation of mouse macrophages was shown indirectly by theingestion of the luminescent agent, lucigenin™, when these cellsphagocytized zymogen particles. FIG. 1 shows that even though untreatedmice had macrophages that were viable and active against this foreignagent, mice treated with B. abortus OPS (5 mg/mouse) had twice thephagocytic activity of controls. FIG. 2 also shows that this doubling ofactivity could be caused by OPS from Y. enterocolitica O:9, a bacteriumthat is unrelated but cross-reacts with Brucella. As noted above, it issuspected that bacterial carbohydrate polymers of unusual conformationor linkage might cause similar immuno-modulation. When the glucosepolymer secreted by Agrobacterium tumefaciens was tested, a dose of 5 mgper mouse caused similar effects, namely a doubling of macrophagephagocytic activity to foreign zymosan particles.

3. Immnuno-Modulation of Humans

Immuno-modulation of humans was shown indirectly by the observation thatan investigator working with these polysaccharides (likely to have beenexposed to trace amounts of these in the past) had exceptionally hightitres to Western Equine Encephalitis vaccine. The investigator had notbeen working with the polysaccharides for a year prior to vaccination.The table below shows an obvious high response to this vaccine. Theaverage titre (not counting investigator #1) was 103±117. Investigator#1 had at least 100-fold more than this.

TABLE 1 Results of Western Equine Encephalitis Vaccination InvestigatorSerum WEE titers #1 Off the scale, greater than 10,000 (worked severaltimes over several years with OPS, likely to have been exposed to OPS orPoly B) #2 320 #3 160 #4 160 #5 Less than 10 #6 Less than 10 #7 20

DISCUSSION

Although the noted sugar is uncommon among the bacterial OPS studied sofar, it does appear to be common in the OPS of a small group of bacteriathat are both invasive and pathogenic. The reason for this correlation(put forward by Cherwonogrodzky et al, 1990) is unknown, but there aretwo possibilities. The first is that the noted sugar has unusualproperties. Although it easily dissolves in water, it is also veryhydrophobic and will sequester into the phenol layer of a phenol-waterextraction. This hydrophobic nature may allow it to insert itself intomammalian membranes, initiating the invasive process, or interfere withthe cell's metabolism in much the same way that hormones attach tomembrane receptors and regulate internal activity. The secondpossibility is that it may interact with the host's internal cellularenzymes, giving the bacterium an advantage for surviving in a normallyhostile environment. As one example, some bacteria and yeasts do notmetabolize unusual amino acid analogues. However, D-tryptophan will bothinhibit growth and greatly enhance toxin expression in Vibrioparahaemolyticus. Possibly if an unusual carbohydrate does not overwhelmthe eucaryotic cell, the cell responds to a metabolic abnormality byinitiating cellular immunity as an automatic defence.

Although the mechanism for this immuno-modulation is being elucidated,the effect of bacterial polysaccharides to enhance cellular immunity isevident. FIGS. 1 to 3 show a doubling of phagocytic activity in responseto a foreign particle. It should be noted that zymosan is an extractfrom yeast and hence an increase in phagocytic activity against thiscompound is likely to indicate an activity against yeast in general.Even before these experiments were done, it was clear that bacterialpolysaccharides had an unusual effect on the macrophages as thesecongregated at the site of bacterial polysaccharide injection when theanimals were sacrificed 1 week later. Practical applications were alsodiscovered accidentally when swine immunized almost 2 years previouslywith B. abortus OPS resisted an outbreak of Haemophilus pleuropneumoniaethat made ill or killed unvaccinated swine.

Although the chemiluminescent assays with mice have given evidence thatsupports the belief that bacterial polysaccharides can act asimmuno-modulators, the accidental outcome in the above noted swinestudy, and the bizarre observation with human vaccination, is far moreencouraging. In the mouse study, high doses of polysaccharides (5 mg ofpolysaccharide per 25 gram mouse) were necessary for enhancing whiteblood cell activity that was only tested 1 week later. In the swinestudy, low doses of polysaccharide (0.1 mg of polysaccharide per 25 kgsow was tested, and lower doses might be equally effective) enhancedtheir immunity which was challenged almost 2 years after treatment. Itshould be noted that in the scientific community it is believed thatswine are closer animal models to humans than are mice. The foregoingstudy has found that bacterial polysaccharides enhance general cellularimmunity and protect swine from 2 dissimilar bacteria, B. suis and H.pleuropneumoniae. Therefore, a further logical embodiment of the presentinvention is that components from one source can be used either before(to protect) or after (as treatment) infections caused by otherbacteria, fungi, yeasts, parasites or viruses (e.g. HIV). It should benoted that in regards to immunity to viruses, an investigator who hasprepared and researched the noted polysaccharides had (as shown in Table1 above) at least 100-fold more anti-WEE antibodies than expected, eventhough the investigator had not worked with the polysaccharides for atleast a year prior to vaccination. Cellular immunity is also importantin clearing cellular abnormalities or pathologies, such as cancer(carcinomas) and the like, and a logical extension of this work is touse bacterial polysaccharides for this purpose.

OPS immuno-modulators of the present invention can be obtained fromBrucella abortus and other cross reactive bacteria. By way of example,such immuno-modulators can be obtained from the group of bacteriaconsisting of Brucella abortus, Brucella suis, Brucella melitensis,Brucella neotomae, Francisella tularensis, Vibrio cholerae, Pseudomonasmaltophila 555, Escherichia coil O:157, H:7, Escherichia coli hermanii,Yersinia enterocolitica O:9, Salmonella landau, and Salmonellagodesberg.

Similarly, Poly B immuno-modulators for the present invention can beisolated from the group consisting of Brucella abortus, Brucella suis,Brucella melitensis, and Agrobacterium tumefaciens.

It has previously been shown that bacterial OPS can be synthesized. Itis therefore an extension of the present invention to provide syntheticcarbohydrates which can replace bacterial OPS as immuno-modulators.

Some strains of Brucella produce both the OPS and the 1,2 beta glucosepolymer (Poly B). As both were shown to enhance mouse white blood cellactivity, there is the logical extension of the present invention thatcombinations of polysaccharides may have synergistic immuno-modulatingeffects.

In a previous study (Cherwonogrodzky et al, 1995), it was found that theOPS could be linked to a carrier (e.g. Lipid A when it formedsmooth-lipopolysaccharide, or sLPS) or used within a delivery system(e.g. liposomes). Bacterial or synthetic polysaccharides are also likelyto have immuno-modulating effects when used in this manner. Accordingly,in another embodiment, the present invention provides the use ofbacterial or synthetic polysaccharides alone, in combination, linked tocarriers or in delivery systems (such as liposomes or microspheres) toenhance general immunity (e.g. against bacteria, viruses, fungi, yeasts,parasites and cellular abnormalities).

CONCLUSIONS

As soluble bacterial polysaccharides were shown to be immuno-modulators(i.e. compounds which enhanced phagocytic activity of macrophages toforeign agents), this contradicts the current scientific view that onlyinsoluble carbohydrates have any such effect. Although the inventors donot limit which bacterial polysaccharides may be effective, they didobserve that polysaccharides consisting of unusual sugars, unusualconformations or unusual linkages, which were unlikely to be readilymetabolized, did enhance the host's cellular activity. They suggest thatwhen the several thousands of bacterial polysaccharides are screened forimmuno-modulating effects, that these characteristics be firstconsidered.

When Brucella OPS was used as a vaccine to protect different animalspecies from brucellosis there appeared to be a wide variation in thedose required for protection: 0.01-0.1 mg for a 25g mouse; 0.1 mg for a25 kg pig; and, 3 mg for a 200 g guinea pig. If humans have immunesystems resembling those of swine rather than rodents, then the effectsof the new immuno-modulators of the present invention are likely to behighly effective, long lasting, effective in low dosages, and to nothave any ill effects.

The present invention also provides for pharmaceutical compositionscontaining the synthetic and non-synthetic bacterial polysaccharides.Further, the polysaccharides of the present invention can beadministered intramuscularly, subcutaneously, intraperitoneally, orally,or by the respiratory route. The polysaccharides can be given before,during, or after vaccination or infection to enhance immunity.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention as outlined in the claims appended hereto.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of treating amammal against an infection caused by a first bacterial species, saidmethod comprising administering intravenously a vaccine comprising anaturally soluble polysaccharide which contains a circular 1,2 β linkedglucose, and has been isolated from a second bacterial species toenhance phagocytic activities of macrophages in said mammal, whereinsaid first bacterial species is from another species than said secondbacterial species, wherein said first bacterial species is selected fromthe group consisting of Brucella and Haemophilus pleurnpneumoniae. 2.The method of treating a mammal against an infection caused by a firstbacterial species, said method comprising administering animmunomodulator comprising a non-phosphorylated naturally solublepolysaccharide isolated from a second bacterial species to enhancephagocytic activities of macrophages in said mammal, wherein said firstbacterial species is from another species than said second bacterialspecies, wherein said polysaccharide is selected from the groupconsisting of an outer-polysaccharide, a Polysaccharide B and apolysaccharide cross-reactive therewith, said polysaccharide containinga 1,2 β linked glucose.
 3. The method of treating a mammal against aninfection caused by a first bacterial species, said method comprisingadministering an immunomodulator comprising a non-phosphorylatednaturally soluble polysaccharide isolated from a second bacterialspecies to enhance phagocytic activities of macrophages in said mammal,wherein said first bacterial species is from another species than saidsecond bacterial species , wherein said outer-polysaccharide comprisesof a polymer of 4-formamido-4,6-dideoxy-D-mannose sugar.
 4. The methodof treating a mammal against an infection caused by a first bacterialspecies, said method comprising administering an immunomodulatorcomprising a non-phosphorylated naturally soluble polysaccharideisolated from a second bacterial species to enhance phagocyticactivities of macrophages in said mammal, wherein said first bacterialspecies is from another species than said second bacterial species,wherein said Polysaccharide B comprises of a polymer of circular 1,2 βlinked glucose.
 5. The method of treating a mammal against an infectioncaused by a first bacterial species, said method comprisingadministering an immunomodulator comprising a non-phosphorylatednaturally soluble polysaccharide which contains a circular 1,2 β linkedglucose, and has been isolated from a second bacterial species toenhance phagocytic activities of macrophages in said mammal, whereinsaid first bacterial species is from another species than said secondbacterial species, wherein said outer-polysaccharide is extracted fromsaid second bacterial species selected from the group consisting ofBrucella abortus, Brucella suis, Brucella melitensis, Brucella neotomae,Yersinia enterocolitica O:9 Francisella tularensis, Vibrio cholerae,Pseudomonas maltophilia 555, Escherichia coli O:15-H:7, Escherichiahermanii, Salmonella landau, Salmonella gosdesberg and a cross reactantthereof.
 6. The method of treating a mammal against an infection causedby a first bacterial species, said method comprising administering animmunomodulator comprising a non-phosphorylated naturally solublepolysaccharide which contains a circular 1,2 β linked glucose, and hasbeen isolated from a second bacterial species to enhance phagocyticactivities of macrophages in said mammal, wherein said first bacterialspecies is from another species than said second bacterial species,wherein said Polysaccharide B is extracted from said second bacterialspecies selected from the group consisting of Brucella abortus, Brucellamelitensis and Agrobacterium tumefaciens.
 7. The method of treating amammal against an infection caused by a first bacterial species, saidmethod comprising administering an immunomodulator comprising anon-phosphorylated naturally soluble polysaccharide which contains acircular 1,2 β linked glucose, and has been isolated from a secondbacterial species to enhance phagocytic activities of macrophages insaid mammal, wherein said first bacterial species is from anotherspecies than said second bacterial species, wherein said first bacterialspecies is selected from the group consisting of Brucella andHaemophilus pleurnpneumoniae.
 8. A method of treating a mammal againstan infection caused by a first bacterial species, said method comprisingadministering intravenously a vaccine comprising a naturally solublepolysaccharide isolated from a second bacterial species to enhancephagocytic activities of macrophages in said mammal, wherein said firstbacterial species is from another species than said second bacterialspecies, wherein said polysaccharide is selected from the groupconsisting of an outer-polysaccharide, a Polysaccharide B and apolysaccharide cross-reactive therewith, said polysaccharide containinga circular 1,2 β linked glucose.
 9. The method of claim 8, wherein saidouter-polysaccharide comprises of a polymer of4-formamido-4,6-dideoxy-D-mannose sugar.
 10. The method of claim 8,wherein said Polysaccharide B comprises of a polymer of 1,2 β linkedglucose.
 11. A method of claim 8, wherein said outer-polysaccharide isextracted from said second bacterial species selected from the groupconsisting of Brucella abortus, Brucella suis, Brucella melitensis,Brucella neotomae, Yersinia enterocolitica O:9 Francisella tularensis,Vibrio cholerae, Pseudomonas maltophilia 555, Escherichia coli O:15-H7,Escherichia hermanii, Salmonella landau, Salmonella gosdesberg and across reactant thereof.
 12. A method of claim 8, wherein saidPolysaccharide B is extracted from said second bacterial speciesselected from the group consisting of Brucella abortus, Brucellamelitensis and Agrobacterium tumefaciens.
 13. A method of treating amammal against an infection caused by a first bacterial species, saidmethod comprising administering an immunomodulator comprising anon-phosphorylated polysaccharide isolated from a second bacterialspecies to enhance phagocytic activities of macrophages in said mammal,wherein said first bacterial species is from another species than saidsecond bacterial species, and said polysaccharide is characterized bybeing soluble in water.
 14. The method of claim 13, wherein saidpolysaccharide is selected from the group consisting of anouter-polysaccharide, a Polysaccharide B and a polysaccharidecross-reactive therewith.
 15. The method of claim 13, wherein saidouter-polysaccharide comprises of a polymer of4-formamido-4,6-dideoxy-D-mannose sugar.
 16. The method of claim 13,wherein said Polysaccharide B comprises of a polymer of circular 1,2 βlinked glucose.
 17. The method of claim 13, wherein saidouter-polysaccharide is extracted from said second bacterial speciesselected from the group consisting of Brucella abortus, Brucella suis,Brucella melitensis, Brucella neotomae, Yersinia enterocolitica O:9Francisella tularensis, Vibrio cholerae, Pseudomonas maltophilia 555,Escherichia coli O:15-H: 7, Escherichia hermannii, Salmonella landau,Salmonella godesberg and a cross reactant thereof.
 18. The method ofclaim 13, wherein said Polysaccharide B is extracted from said secondbacterial species selected from the group consisting of Brucellaabortus, Brucella melitensis and Agrobacterium tumefaciens.
 19. Themethod of claim 13, wherein said first bacterial species is selectedfrom the group consisting of Brucella and Haemophilus pleuropneumoniae.20. A method of treating a mammal against an infection caused by a firstbacterial species, said method comprising administering intravenously avaccine comprising a polysaccharide which contains a circular 1,2 βlinked glucose, and has been isolated from a second bacterial species toenhance phagocytic activities of macrophages in said mammal, whereinsaid first bacterial species is from another species than said secondbacterial species, and said polysaccharide is characterized by beingsoluble in water.
 21. A method of claim 20, wherein said first bacterialspecies is selected from the group consisting of Brucella andHaemophilus pleuropneumoniae.
 22. A method of claim 20, wherein saidpolysaccharide is selected from the group consisting of anouter-polysaccharide, a Polysaccharide B and a polysaccharidecross-reactive therewith.
 23. The method of claim 22, wherein saidouter-polysaccharide comprises of a polymer of4-formamido-4,6-dideoxy-D-mannose sugar.
 24. The method of claim 22,wherein said Polysaccharide B comprises of a polymer of 1,2 β linkedglucose.
 25. A method of claim 22, wherein said outer-polysaccharide isextracted from said second bacterial species selected from the groupconsisting of Brucella abortus, Brucella suis, Brucella melitensis,Brucella neotomae, Yersinia enterocolitica O:9 Francisella tularensis,Vibrio cholerae, Pseudomonas maltophilia 555, Escherichia coli O:15-H:7,Escherichia hermannii, Salmonella landau, Salmonella godesberg and across reactant thereof.
 26. A method of claim 22, wherein saidPolysaccharide B is extracted from said second bacterial speciesselected from the group consisting of Brucella abortus, Brucellamelitensis and Agrobacterium tumefaciens.